DESCRIPTION (Verbatim from the application): In the heart and peripheral vascular diseases, angiogenesis can improve blood flow and facilitate long-term survival of ischemic tissues. This complex process involves endothelial cell (ECs) and vascular smooth muscle cell (VSMC) adhesion, migration, proliferation and differentiation. Humoral agents such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been suggested as mediators of this process. However, vessels are also richly innervated with sympathetic nerves, which are known to contain trophic factors. Recently, we discovered that neuropeptide Y (NPY), a sympathetic transmitter stimulates angiogenesis in vitro and in vivo, with potency and efficacy comparable to those of bFGF or VEGF. In addition, human ECs possess a complete NPY autocrine system, which synthesizes NPY and its receptors (Rs; Y1-Y5)as well as the "converting enzyme," dipeptidyl peptidase IV (DPPIV). In the last two years we made a major discovery that in the in vivo rat angiogenic model, femoral artery occlusion and hence limb ischemia results in substantial NPY release from the ischemic leg yet no NPY Rs are detected, thus preventing NPY from exerting its angiogenic activity. In a follow up study, we demonstrated that local NPY administration, both up-regulates NPY Rs and restores capillary density in the skeletal muscles to the level observed in the non-ischemic leg. Based on these exciting discoveries, we now propose to establish if NPY constitutes a new endogenous angiogenic system stimulated by ischemia, and to determine the subtypes of angiogenic Rs and mechanisms of NPY action. To this end, two ischemic models are proposed: the hindlimb (rat and mouse) and the heart (rat). Specific aims are to determine: 1) if ischemia/hypoxia up-regulates NPY release and the expression of its Rs in spatial and temporal relation to angiogenesis in the ischemic tissues, and in vitro in ECs; 2) the mechanisms of NPY's angiogenic effects and its dependence on DPPIV, and interactions with other growth factors: (bFGF, VEGF, nitric oxide); 3) which of the five NPY Rs are angiogenic; and 4) if the specific NPY R agonist revascularizes the ischemic heart and hindlimb. We will focus on the Y2 and Y5 subtypes, which are suggested by our recent findings, but the role of the predominant vascular NPY R, the Yl, will also be investigated. These studies will be performed in rats treated with NPY (or other factors) in slow release pellets or over-expressing the NPY gene, and in NPY, Yl, Y2, and Y5 knockout (KO) mice. The angiogenic A(s) will be determined by studying their mRNA and protein expression using KO mice and/or specific NPY R agonists or antagonists in the above ischemic models, and in the in vitro angiogenic assays. NPY's angiogenic activity will be assessed structurally (e.g. by angiograms, tissue pathology, capillary/fiber ratio, and mitotic index) and functionally (by regional blood flow, cardiac output and motor scoring scale). This research will be the first to establish the role of NPY in ischemia-driven angiogenesis, and to determine the specific NPY Rs mediating this action. In addition to providing an understanding of the NPY's role as a mediator of neurogenic angiogenesis, these findings will open new avenues for treatment of ischemic diseases.